Abstract: F420-dependent glucose-6-phosphate dehydrogenase (FGD), is an essential enzyme found in Mycobacterium tuberculosis, the causative agent of tuberculosis disease. The reaction catalyzed by FGD is the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone. The substrate, G6P donates electrons for the reduction of the F420 Cofactor. The initial goal of this study is to characterize the hydride transfer reaction mechanism of FGD using steady state and pre-steady state kinetic methods. Our preliminary kinetic studies with wtFGD, includes a series of experiments that address this goal. Specific aim 1 proposes to use site-directed mutagenesis, along with binding studies, steady-state and pre-steady state kinetic experiments as a means to investigate the functionality of conserved active site amino acid residues. We have recently solved the crystal structure of FGD in complex with its substrate, G6P. This crystal structure along with a previously published structure implicates several conserved active site residues that may participate within the hydride transfer reaction of FGD. However, the ability to interrogate these amino acids via kinetics has been limited until now. The goal of this aim is to provide experimental data to elucidate the mechanism of FGD. The PI's laboratory has already created several of these FGD variants, one of which is H40A. A concurrent goal is to conduct cyclic voltammetry experiments in order to observe the effects of active site mutations on the potential of the F420 Cofactor. Specific aim 2 is to investigate hydride tunneling within FGD. The study of hydride tunneling, in enzymes has become more prevalent in recent years and has been suggested that this is a catalytic strategy for enzymes that drive hydrogen transfer reactions. Generally speaking, investigations of this type have not been pursued for F420 Cofactor dependent enzymes. The goal of specific aim 2 is to use isotopic labeling, pre-steady state and steady-state kinetic analysis to study hydride tunneling within FGD. Specific aim 3 is to utilize solvent isotope effects experiments to conduct a proton inventory to determine the number of exchangeable protons within FGD that are active. In summation, this proposal describes fundamental studies to elucidate the kinetics and mechanism of an important naturally occurring tuberculosis enzyme, but one for which relatively little such data are currently available.